Antimicrobial compounds

ABSTRACT

This invention provides compounds and methods for treating, with said compound, a mycobacterial infection by administering to an animal a pharmaceutical composition containing a compound having the formula R—SO n -Z-CO—Y, where R is an alkyl groups having 6-20 carbon atoms, unsaturated hydrocarbon groups having 6-20 carbon atoms, or alkyl groups having 6-20 carbon atoms interrupted by at least one aromatic ring; Z is —CH 2 —, —CH 2 CH 2 —, —NH—NH—, —O—, ——NH—, —O—NH—, —CH 2 —NH—, —CH 2 —O—, —NH—O—, —NH—CH 2 —, —O—CH 2 —, and —CH═CH—; Y is —NH 2 , —O—CH 2 —C 6 H 5 , —CO—CO—O—CH 3 , and —O—CH 3 ; and n is 1 or 2. It has been discovered that these compounds treat microbially-based infections caused by corynebacteria, nocardiae, rhodococcus, and mycobacteria. These compounds may be used to treat mycobacterial cells, such as  Mycobacteria tuberculosis , drug resistant  M. tuberculosis, M. avium intracellulare, M. leprae, M. paratuberculosis , and pathogenic  Mycobacteria  sp.

CROSS-RELATED AND PRIORITY APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/486,550, filed Aug. 28, 2000, now U.S. Pat. No. 6,713,654, whichis the National Phase under 35 U.S.C. § 371 of PCT/US98/17830, filedAug. 28, 1998, which claims benefit of priority of U.S. application Ser.No. 60/056,272, filed Aug. 29, 1997. These priority documents are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the synthesis and in vivo application ofcompounds which have antibiotic activity against microbes thatsynthesize mycolic acid, including Mycobacterium sp., particularly drugresistant Mycobacterium strains, and to the use of these compounds totreat any susceptible pathogenic microorganism or parasite.

2. Review of Related Art

The emergence of multiply drug resistant (MDR) strains of Mycobacteriumtuberculosis and other atypical mycobacteria which infectimmunocompromised patients (e.g., AIDS patients) highlights the need forcontinued antibiotic development.

Mycobacterium sp. synthesize a multitude of complex lipids andglycolipids unique to this genus, making these biochemical pathwaysattractive targets for drug therapy (Bloch, K., “Control mechanisms forfatty acid synthesis in Mycobacterium smegmatis,” Adv. Enzymol. 45:1-84,1977; Brennan, P. J., and Nikaido, H., “The envelope of mycobacteria,”Ann. Rev. Biochem. 64:29-63, 1995). The β-ketoacyl synthase (KS) ofparticulate Type II fatty acid synthases or the corresponding domain ofthe polyfunctional Type I fatty acid synthases catalyzes the criticaltwo-carbon homologation during buildup of the growing fatty acid chain.This process typically gives acids of length C₁₆ to C₁₈. In chainelongation of normal fatty acids, carried out for example bymycobacteria, CoA and/or acyl-carrier protein (ACP) thioesters of theseacids are further reacted with malonyl-CoA to greatly extend theirlength to 60-90 carbons. These high molecular weight acids are knowncollectively as mycolic acids.

Mycolic acids are a group of complex, long, branched chain fatty acidsthat are vital for the growth and survival of mycobacteria. Mycolicacids comprise the single largest component of the mycobacterial cellenvelope. Little is known about the nature of the biosynthetic enzymesinvolved, but evidence suggests some similarity to conventional fattyacid synthases (Bloch, 1977; Brennan, et al., 1995). These unusuallylong lipid molecules form a waxy coat of limited permeability.

The presence in mycobacteria of particular modified fatty acids havingcomplex and well-organized structures presents a potentially attractivetarget for drug design (Young, D. B., and Duncan, K., “Prospects for newinterventions in the treatment and prevention of mycobacterial disease,”Ann Rev. Microbiol. 49:641-673, 1995). It has been suggested thatisoniazid inhibits mycolic acid synthesis as its potential mechanism ofaction (Takayama, K., Wang, L., and David, H. L., “Effect of isoniazidon the in vivo mycolic acid synthesis, cell growth, and viability ofMycobacterium tuberculosis,” Antimicrob. Agents Chemother., 2:29-35,1972; Takayama, K., Schnoes, H. K., Armstrong, E. I., and Booyle, R. W.,“Site of inhibitory action of isoniazid in the synthesis of mycolicacids in Mycobacterium tuberculosis,” J. Lipid Res., 16:308-317, 1975;Quemard A., Dessen A., Sugantino M., Jacobs W. R., Sacchettini J. C.,Blanchard J. S. “Binding of catalase peroxide-activated isoniazid towild-type and mutant Mycobacterium tuberculosis enoyl-ACP reductases,”J. Am. Chem. Soc., 118:1561-1562, 1996; Baldock C., Rafferty J. B.,Sedenikova S. E., Baker P. J., Stuitje A. R, Slabas A. R., Hawkes T. R.,Rice D. W. “A mechanism of drug action revealed by structural studies ofenoyl reductase,” Science, 274:2107-2110, 1996; Quemard A., SacchettiniJ. C., Dessen A., Vilcheze C., Bittman R., Jacobs W. R., Blanchard J.S., “Enzymatic characterization of the target for isoniazid inMycobacterium tuberculosis, Biochemistry, 34:8235-8241, 1993; Msluli,K., D. R. Sherman, M. J. Hickey, B. N. Kreiswirth, S. Morris, C. K.Stover, and C. E. Barry, III, “Biochemical and genetic data suggest thatInhA is not the primary target for activated isoniazid in Mycobacteriumtuberculosis,” J. Infect. Dis., 174:1085-1090, 1996; Dessen A., A.Quemard, J. S. Blanchard, W. R. Jacobs, and J. C. Saccettini, “Crystalstructure and function of the isoniazid target of Mycobacteriumtuberculosis,” Science, 267:1638-1641, 1995; Banerjee, A., E. Dubnau, A.Quemard, V. Balasubramanian, K. S. Um, T. Wilson, D. Collins, G.deLisle, W. R. Jacobs, Jr., “InhA, a gene encoding a target forisoniazid and ethionamide in Mycobacterium tuberculosis.” Science,263:227-230, 1994). This finding might be expected to stimulate a searchfor novel compounds that act upon the lipid synthetic pathways ofmycobacteria as a fresh approach for antibiotic development.Surprisingly, however, lipid biosynthesis has not been exploited fordrug development in these organisms. No drugs which specifically inhibitmycobacterial lipid synthesis have been developed other than isoniazid,and there remains a need for new drugs to treat the growing problem ofmulti-drug resistant mycobacteria.

SUMMARY OF THE INVENTION

This invention is directed to novel compounds having antimicrobialactivity, particularly antimicrobial effectiveness against multi-drugresistant mycobacteria.

This invention is also directed to a method for treating mycobacterialinfection by drug resistant strains through use of independenttherapeutic targets.

These and other objects of the invention are achieved by one or more ofthe following embodiments. In one embodiment, this invention provides acompound having the formula: R—SO_(n)-Z-CO—Y, where R is preferably analkyl group having 6-20 carbons; Z is preferably a radical selected from—CH₂—, —O—, and —NH—, two of these radicals coupled together or—CH₂═CH₂—; Y is preferably —NH₂, —O—CH—C₂H₆ —CO—CO—O—CH₃, or —O—CH₃; andn is 1 or 2. In particularly preferred embodiments. R is a branchedalkyl group, or R is a linear alkyl group interrupted by an aromaticring.

In another embodiment, this invention provides a method of inhibitinggrowth of a microbial cell which synthesizes α-substituted, β-hydroxyfatty acids. The method comprising treating the cell with a compoundhaving the formula: R—SO_(n)-Z-CO—Y, as described above. In particular;cells inhibited by the compound of this invention are cells whichsynthesize α-substituted, β-hydroxy fatty acids selected from the groupconsisting of corynemycolic acid, nocardic acid, and mycolic acid.Preferably, the method is used to inhibit growth of microbial cellsselected from the group consisting of corynebacteria, nocardiae,rhodococcus, and mycobacteria. More preferably, the method is used toinhibit growth of mycobacterial cells, such as Mycobacteriumtuberculosis, drug-resistant M. tuberculosis, M. avium intracellulare,M. leprae, or M. paratuberculosis.

In yet another embodiment, this invention provides a method for treatinga mycobacterial infection by administering to an animal a pharmaceuticalcomposition containing a compound having the formula: R—SO_(n)-Z-CO—Y,as described above.

The present inventors have synthesized and tested a number of sulfonesand sulfoxides having structures based upon the reaction intermediatesof the β-ketoacyl synthase reaction of fatty acid synthase. A .number ofthese compounds have demonstrated in vitro activity against virulent M.tuberculosis (see Table 1). The desireable characteristics found amongthe compounds tested included: potency, in vivo activity,reproducibility of MIC data, ease of synthesis, and chemical stability.Use of the compounds of this invention in drug therapy against multiplydrug resistant tuberculosis will provide a means to treat both patientspresently suffering from active disease, and the millions of potentialpatients who harbor quiescent disease which may become active as aresult of immunosuppression or other systemic disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of the β-ketoacyl synthasereaction.

FIG. 2 shows two-dimensional thin layer chromatography of mycolic acidsextracted from M. avium-intracellulare (Left Panel) and the same extractfollowing treatment with n-octanesulphonylacetamide (Right Panel).

FIGS. 3A and 3B show photomicrographs of M. bovis BGC with (B) orwithout (A) prior n-octanesulphonylacetamide treatment.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The compounds synthesized according to this invention are based ontheoretical transition-state intermediates of the β-ketoacyl synthasereaction of fatty acid synthase (E.C. 2.3.1.85). The β-ketoacyl synthasereaction is common to fatty acid synthesis in prokaryotes andeukaryotes, including Mycobacteria. The family of compounds shown inTable 1 was found to be cytotoxic against a variety of mycobacteria,including drug resistant strains. Instead of inhibiting Type I/Type IIfatty acid synthesis in mycobacteria, however, Compound HIII-50 (III-50)inhibits the synthesis of mycolic acids, specialized fatty acids foundparticularly in Mycobacteria sp. One of the key steps in mycolic acidsynthesis is fatty acid elongation, which employs carbon-carboncondensation similar to the β-ketoacyl synthase reaction in de novofatty acid synthesis. It is believed that these compounds act at thisfatty acid elongation step critical to mycolic acid synthesis.

The transition states for all of the two-carbon elongation reactions arepresumed, on existing biochemical evidence and mechanistic grounds, tobe quite similar (see FIG. 1) and involve acylanion formation bydecarboxylation of malonyl-CoA/ACP to provide a nucleophile (FIG. 1,step A) to react with the thioester-bound acyl chain to generatetetrahedral intermediate (FIG. 1, step B). This transient intermediateis anticipated to collapse (FIG. 1, step C) to extend the growing acylunit (RCO—) by two carbons. Further steps then reduce the β-keto groupof the ACP/CoA derivative and finally return it to step A to begin thechain extension cycle again.

Low molecular weight organic compounds which are potential mimics of thetetrahedral intermediate (see stepB) may be expected to be inhibitors ofthis biochemical process, and such compounds are represented by FormulaI.R—SO_(n)-Z-CO—Y,  Formula Iwherein R is hydrocarbon, such as an alkyl group;

-   -   n is 1 or 2;    -   Z is a hydrocarbon linking moiety that may contain a heteroatom;        and    -   Y is a hydrocarbon end group moiety that may contain one or more        heteroatoms.

In preferred compounds according to Formula I, R may be an n-alkyl grouphaving 6-20 carbons, preferably 8, 10 or 12 carbons, and may besaturated or unsaturated, branched or unbranched, or the alkyl chain maybe interrupted by a aromatic ring to give ortho-, meta-, orpara-disubstitution. Z is preferably —CH₂—, —CH₂—CH₂—, cis or trans—CH═CH—, —NH—CH₂—, —CH₂—NH—, —O—CH₂—, or —CH₂—O—, and Y is preferably—NH₂, —O—CH₂—C₆H₅, —CO—O—CH₃ —CO—CO—O—CH₃ or —O—CH₃. Between R and Z isa sulfur atom in the form of a sulfoxide or sulfone. The sulfone III-50and close structural analogues with shorter and longer saturated andunsaturated alkyl side chains, as well as alternative mimics of thetetrahedral intermediate of step B are exemplified but not exclusivelyrepresented by the structures shown in Table 1.

The compounds of Formula I may be synthesized by a variety of routesincluding alkylation of a mercaptoester to produce a thioether (see,e.g., R. L. Smith et al., J. Med. Chem., 1977, 20:540-547), followed byoxidation to afford a sulfoxide or sulfone and conversion of the esterto an amide with ammonia or a substituted amine. A suitable syntheticscheme is shown in Scheme 1 and exemplified in Example 1.

Alternatively, a sulfonate salt, for example the sodium salt, may bereacted with a haloester to give a thioether (i.e.R—SO₂—Na⁺+X—(CH₂)_(n)—COOR′→R—SO₂—(CH₂)_(n)—COOR′; see, e.g., E.Gipstein, C. G. Willson and H. S. Sachdev, J. Org Chem., 1980,45:1486-1489), which can be oxidized and ammonolyzed to thecorresponding amide as above. Typically, X═Cl, Br or I. A secondalternative would be reaction of a haloamide (X—(CH₂)_(n)—CONH₂) witheither a sulfonate salt, as above, or with a thiolate anion followed byoxidation to similarly yield the sulfonyl amide or the sulfoxide amide(see, e.g., S. Huenig and O. Boes, Liebigs Annalen der Chemie, 1953,579:23-26). It will be apparent to the skilled worker that the sense ofthese reactions can be reversed so that a halohydrocarbon may be reactedwith a mercaptoamide or the salt of a sulfenylamide or sulfenylester toobtain the same products. A third alternative would be reaction of athiol (R—SH) with propiolate ester or amide to form a sulfanyl-acrylicester or amide followed by oxidation to the sulfoxide or sulfone.Finally, a halo- or mercaptonitriles can be reacted by the above schemesto give thioethers or sulfonylnitriles, which can be hydrolyzed to theamides. Where other alternative synthetic routes to produce thecompounds of Formula I occur to the skilled worker, the products of suchsynthesis are also within the contemplation of this invention.

The compounds according to Formula I may be used as antibiotics againstmicrobes having in their cell walls α-substituted, β-hydroxy fattyacids, such as corynemycolic acids(e.g., C30), nocardic acids (e.g.,C50) or mycolic acids (e.g., C90). Unless otherwise indicated, use ofthe term mycolic acids herein refers to any of these long chainα-substituted, β-hydroxy fatty acids. In particular, compounds accordingto Formula I exhibit antibiotic activity against corynebacteria,nocardiae, rhodococcus and mycobacteria. More particularly, thesecompounds are effective against Mycobacterium tuberculosis,drug-resistant M. tuberculosis, M. avium intracellulare, M. leprae, orM. paratuberculosis.

Preferred compounds according to Formula I will have substantialantibiotic activity against susceptible organisms (see, e.g., Table 1).Antibiotic effectiveness of compounds according to Formula I may bedetermined as described below or by use of assays described in U.S. Pat.No. 5,614,551, which is incorporated herein by reference. In particulr,U.S. Pat. No. 5,614,551 describes an in vitro therapeutic index based oncomparison of the concentration which inhibits growth of normalfibroblasts to the minimal inhibitory concentration for a compound, andpreferred compounds will have an in vitro therapeutic index of at least2, more preferably at least 5, and most preferably at least 10.

Novel drug therapy, using compounds of this invention which areeffective against multiply drug resistant tuberculosis, will aid intreating both patients presently suffering from active disease, and themillions of potential patients who harbor quiescent disease which maybecome active as a result of immunosuppression or other systemicdisease. These drugs will also be useful against the, “atypicalmycobacteria” such as M. avium-intracellulare, a common AIDS pathogen,and other species that are commonly drug resistant. Given thebiochemical similarity between M. tuberculosis and M. leprae, thesedrugs may be expected to be useful in the treatment of leprosy (Hansen'sdisease). Potential use in livestock or other veterinary applicationsinclude treatment of infections by Mycobacterium paratuberculosis, alsoknown as Johne's bacillus, an organism that produces a chronic enteritisin ruminants (e.g., cattle and sheep) which is invariably fatal, andRhodococcus, another organism which produces mycolic acids as well aspotentially fatal respiratory infections in horses and immunocompromisedpatients. Treatment of human patients infected with M. paratuberculosisis also within the contemplation of this invention.

Treatment according to this invention involves administering thecompound of Formula I to the subject of treatment. Pharmaceuticalcompositions containing any of the compounds of this invention may beadministered by parenteral (subcutaneously, intramuscularly,intravenously, intraperitoneally, intrapleurally, intravesicularly orintrathecally), topical, oral, rectal, or nasal or inhalation route, asnecessitated by choice of drug, pharmaceutical carrier, and disease.

Therapeutic compounds according to this invention are preferablyformulated in pharmaceutical compositions containing the compound and apharmaceutically acceptable carrier. The Concentrations of the activeagent in pharmaceutically acceptable carriers will depend onsolubilities. The dose used in a particular formulation or applicationwill be determined by the requirements of the particular type of diseaseand the constraints imposed by the characteristics and capacities of thecarrier materials. The pharmaceutical composition may contain othercomponents so long as the other components do not reduce theeffectiveness of the compound according to this invention so much thatthe therapy is negated. Pharmaceutically acceptable carriers are wellknown, and one skilled in the pharmaceutical art can easily selectcarriers suitable for particular routes of administration (see, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985).

Dose and duration of therapy will depend on a variety of factors,including the therapeutic index of the drugs, disease type, patient age,patient weight, and tolerance of toxicity. Dose will generally be chosento achieve serum concentrations from about 1 ng to about 100 μg/ml,typically 0.1 μg/ml to 10 μg/ml. Preferably, initial dose levels will beselected based on their ability to achieve ambient concentrations shownto be effective in in vitro models and in vivo models and in clinicaltrials, up to maximum tolerated levels. The dose of a particular drugand duration of therapy for a particular patient can be determined bythe skilled clinician using standard pharmacological approaches in viewof the above factors. The response to treatment may be monitored byanalysis of blood or body fluid levels of the compound according to thisinvention, measurement of activity of the compound or its levels inrelevant tissues or monitoring disease state in the patient. The skilledclinician will adjust the dose and duration of therapy based on theresponse to treatment revealed by these measurements.

Typically, the compositions described above will be combined or usedtogether or in coordination with one or more other therapeuticsubstances, e.g., other drugs presently used in treating tuberculosisThe compound of Formula I, or a synergistic combination of inhibitors,will of course be administered at a level (based on dose and duration oftherapy) below the level that would kill the animal being treated.Preferably, administration will be at a level that will not irreversiblyinjure vital organs, or will not lead to a permanent reduction in liverfunction, kidney function, cardiopulmonary function, gastrointestinalfunction, genitourinary function, integumentary function,musculoskeletal function, or neurologic function. On the other hand,administration of inhibitors at a level that kills some cells which willsubsequently be regenerated (e.g., endometrial cells) is not necessarilyexcluded.

EXAMPLES

In order to facilitate a more complete understanding of the invention, anumber of Examples are provided below. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only.

Example 1 Synthesis of Substitued Sulfonylamides

To illustrate the synthetic method shown in Scheme 1, a 25 g synthesisof III-50 was carried out.

Methyl n-Octylthioacetate. Octyl bromide (50.21 g., 0.26 mole), methylthioglycoate (22.35 ml, 26.53 g, 0.25 mole) and potassium carbonate(34.5 g, 0.25 mole) were charged into a 1 L round-bottomed flask. Tothis mixture was added 350 ml of acetone and the suspension was refluxedfor 48 h. After cooling to room temperature, the reaction mixture wasfiltered with the aid of acetone. The filtrate was evaporated underreduced pressure and the residue thus obtained was purified bydistillation under reduced pressure. The fraction distilling at 118-121°C./3.8 mm Hg was collected. Yield: 48 g; 88%

IR (neat): 2924, 1736, 1435, 1278, 1133, 1011 cm¹. ¹H NMR (CDCl₃): δ3.64 (s, 3H), 3.13 (s, 2H), 2.52 (t, 2H, J=7.2 Hz), 1.47 (m, 2H),1.2-1.4 (m, 10H), 0.88 (t, 3H, J=6.8 Hz). ¹³C, NMR: (CDCl₃): δ 13.9,22.5, 28.6, 29.0, 29.02, 31.6, 32.5, 33.3, 52.1, 171.0.

Methyl n-Octanesulphonylacetate. A 3 L three-necked round-bottomed flask(fitted with a mechanical stirrer) was charged sequentially withammonium heptamolybdate tetrahydrate (56 g, 0.045 mole), methyln-octylthioacetate (40 g, 0.183 mole) and 1.5 L absolute alcohol(Schultz, H S, Freyermuth, H B., and Buc, S R., J. Org. Chem.28:1140-1142, 1963). the vigorously stirred solution was cooled to 0° C.and to this cooled solution was added 104 ml of 30% hydrogen peroxidesolution (0.732 mole) over a period of 1 h. The reaction mixture wasallowed to warm to room temperature over a period of 2 h and thenstirred for another 24 h when thin layer chromatography on silica showedcomplete disappearance of the starting material. The reaction mixturewas filtered and the filtrate was evaporated under reduced pressure. Theresidue thus obtained was dissolved in ethyl acetate (1 L) and washedwith water (100 ml×2), brine (100 ml). The organic layer was dried overanhydrous MgSO₄ and, after filtration of the solvents, was evaporatedunder reduced pressure to obtain the sulfone as a waxy solid, 36 g, 78%.The produce appeared to be pure (>95%) by NMR and was submitted to thenext reaction without further purification.

IR (neat): 2919, 2848, 1743, 1460, 1437, 1329, 1278, 1218, 1137, 1108,1010, 912, 723 cm⁻¹. ¹H NMR (CDCl₃): δ 3.96 (s, 2H), 3.79 (s, 3H), 3.21(t, 3H, J=8 Hz), 1.80 (m, 2H), 1.20-1.45 (m, 10H), 0.81 (t, 3H, J=6.7Hz). ¹³C NMR (CDCl₃): δ 13.9, 21.7, 22.4, 28.2, 28.7, 28.8, 31.5, 53.1,53.4, 57.0, 163.4.

n-Octanesulphonylacetamide. A solution of methyln-octanesulphonylacetate (35 g, 0.143 mole) in 350 ml of anhydrousmethanol was stirred magnetically at room temperature. To this solutionwas added 24 ml of aqueous ammonium hydroxide (27%, 6.48 g, 0.185 mole)in drops over a period of 30 minutes. The solution was stirred for 24 hand the white precipitate formed was filtered. The solid wasrecrystallized from hot ethyl acetate to obtain the required acetamideIII-50 as a crystalline solid, mp. 140-142° C., 33 g, 97.6%.

IR (neat): 3386, 2920, 1659, 1420, 1315, 1286, 1129, cm⁻¹. ¹H NMR(CDCl₃): δ 6.56 (br s, 1H, NH), 5.69 (br s, 1H, NH) 3.86 (s, 2H), 3.14(app 1:1:1 triplet, J app=8.0 Hz, 2H), 2.16 (m, 2H), 1.86 (m, 2H),1.1-1.3 (m, 12H), 0.86 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃COCD₃): δ 14.7,22.9, 23.6, 29.4, 30.1, 30.15, 30.8, 32.8, 54.0, 164.8 HRMS calculatedfor C₁₀H₂₅N₂O₃S (M+NH₄ ⁻) 253.1586, found 253.1587.

Example 2 Synthesis of Substituted Sulfonylamides

To illustrate the synthetic methods shown in Scheme 2 below, thefollowing syntheses of compounds I-31 and I-89 were carried out.

(E/Z)-3-decylsulfanyl-acrylic acid methyl ester (1): Triethylamine (0.5mL, 3.6 mmol) was added dropwise to a solution of decanethiol (2.5 mL,12.1 mmol) and methyl propiblate (4.3 mL, 48.3 mmol) in dichloromethane(20 mL). The solution was stirred at room temperature for 20 min, underargon, after which it was diluted with water (60 mL) and extracted withdichloromethane (3×60 mL). The organic layer was washed with water (2×60mL), brine (60 mL), dried over anhydrous magnesium sulfate, andconcentrated in vacuo. Purification by flash chromatography (silica gel,ethyl acetate:hexanes 1:49) yielded a clear oil (3.06 g, 98%) as amixture of isomers (E:Z, 6:1), which was carried through to the nextstep.

E isomer: IR (neat, cm⁻¹): 2920, 2848, 1712, 1580, 1460, 1430, 1300,1255, 1215, 1160, 1041, 1015, 945, 827, 715, 697. ¹H NMR (CDCl₃) δ 0.88(t, 3H, J=6.7 Hz), 1.26 (bs, 12H), 1.40 (m, 2H), 1.67 (quint, 2H, J=7.3Hz), 2.78 (t, 2H, J=7.4 Hz), 3.72 (s, 3H), 5.74 (d, 1H, J=15.2 Hz), 7.70(d, 1H, J=15.2 Hz). Z isomer: IR (neat, cm⁻¹): 2920, 2848, 1712, 1580,1460, 1430, 1300, 1255, 1215, 1160, 1041, 1015, 945, 827, 715, 697. ¹HNMR (CDCl₃) δ 0.88 (t, 3H, J=6.7 Hz), 0.9-1.9 (app. s & several m, 16H),2.77 (t, 2H, J=7.4 Hz), 3.75 (s, 3H), 5.85 (d, 1H, J=10.2 Hz), 7.1 (d,1H, J=10.2 Hz).

(E)-3-decylsulfanyl-acrylic acid methyl ester (2): Compound 1 (0.50 g,1.93 mmol), was dissolved in methanol (8.0 mL), and cooled to 0° C.,added to a solution of oxone (1.84 g, 6.0 mmol) in water (8.0 mL, 49.5%)at 0° C., and stirred for 4 h at room temperature. The reaction was thendiluted with water (60 mL), and extracted with chloroform (3×60 mL). Theorganic extract was washed with water (2×60 mL), brine (60 mL), driedover anhydrous magnesium sulfate, and concentrated in vacuo.Purification by flash chromatography (silica gel, ethyl acetate:hexanes1:1) yielded white crystals (0.53 g, 93%) as a mixture of isomers (E:Z,6:1). Purification by preparative TLC (ethyl acetate:hexanes 1:1)yielded compound 2 as white crystals.

mp 62-63° C.; IR (CH₂Cl₂, cm⁻¹): 3060, 2950, 2920, 2845, 1730, 1470,1435, 1280, 1230, 1165, 1130, 995, 965, 815, 765, 690, 573. ¹H NMR(CDCl₃) δ 0.88 (t, 3H, J=6.7 Hz), 1.27 (bs, 12H), 1.43 (m, 2H), 1.80(sym. m, 2H), 3.05 (sym. m, 2H), 3.86 (s, 3H), 6.88 (d, 1H, J=15.3 Hz),7.34 (d, 1H, J=15.3 Hz). Calculated for C₁₄H₂₆O₄S: C, 57.90, H, 9.02, S,11.04. Found: C, 57.94, H, 9.12, S, 11.16.

Propiolamide (3): Methyl propiolate (2.0 mL, 22.5 mmol) was added toliquid ammonia at −78° C. and stirred for 2 hours. Evaporation at roomtemperature yielded compound 3 as white crystals (1.46 g, 94%).

mp 58-61° C. (lit.[5], 61-62° C.); ¹H NMR (D₂O) δ 3.50 (s, 1H), 4.43(bs, 2H).

(E/Z)3-decylsulfanyl-propionamide (4): Triethylamine (0.5 mL, 3.6 mmol)was added dropwise to a solution of decanethiol (1.36 mL, 6.56 nmol) andpropiolamide (1.80 g, 26.1 mmol) in dichloromethane (20 mL). Thesolution was stirred at room temperature for 20 min under argon, afterwhich it was diluted with water (60 mL) and extracted withdichloromethane (3×60 mL). The organic layer was washed with water (2×60mL), brine (60 mL), dried over anhydrous magnesium sulfate, andconcentrated in vacuo. Purification by flash chromatography (silica gel,ethyl acetate:hexanes 3:1),yielded compound 4 as white crystals (1.45 g,91%) as a mixture of isomers (E:Z, 1:10), which was carried through tothe next step.

Mixture of isomers: mp 65-70° C.,; IR (CH₂Cl₂, cm⁻¹): 3400, 3330, 3280,3200, 2920, 2840, 1725, 1650, 1570, 1460, 1400, 1300, 1190, 770.E-isomer: ¹H NMR(CDCl₃) δ 0.89 (t, 3H, J=6.8 Hz), 1.27 (bs, 12H), 1.41(m, 2H), 1.70 (m, 2H), 2.74 (t, 2H, J=7.5 Hz), 5.4 (bs, 2H, NH₂), 5.82(d, 1H, J=10.1 Hz), 6.95 (d, 1H, J=10.1 Hz).

(E)-3-decylsulfonyl-propionamide (5): Compound 4 (0.50 g, 2.05 mmol) wasdissolved in methanol (8.0 mL), cooled to 0° C. and added to a solutionof oxone (1.84 g, 6.0 mmol) in water. (8.0 mL, 49.5%) at 0° C. andstirred for 4 h at room temperature. The solution was then diluted withwater (60 mL), and extracted with chloroform (3×60 mL). The organicextract was washed with water (2×60 mL), brine (60 mL), dried overmagnesium sulfate, and concentrated in vacuo. Purification by flashchromatography (silica gel, ethyl acetate:hexanes 17:3) yielded compound5 as white crystals (0.52 g, 92%) as a mixture of isomers (E:Z, 6:1).Purification by preparative TLC (ethyl acetate:hexanes 3:1) yieldedcompound 5 as white crystals.

mp 148-149° C.; IR (CHCl₃, cm⁻¹): 3390, 3140, 3060, 2920, 2840, 1700,1615, 1455, 1395, 1320, 1130, 960, 814. ¹H NMR (CDCl₃) δ 0.089 (t, 3H,J=6.8 Hz), 1.27 (bs, 12H), 1.43 (m, 2H), 1.80 (m, 2H) 3.05 (sym. m, 2H),5.67 (bs, 1H, NH), 5.85 (bs, 1H, NH), 6.94 (d, 1H, J=14.8 Hz), 7.35 (d,1H, J=14.8 Hz). Calculated for C₁₃H₂₅NO₃S: C, 56.70, H, 9.15, N 5.09, S11.64. Found: C, 56.78, H, 9.16, N, 5.04, S, 11.76.

Example 3 In vitro Activity of Sulfones and Sulfoxides AgainstMycobacteria

Various compounds were tested as described in U.S. Pat. No. 5,614,551 todetermine minimum inhibitory concentratios (MIC) of the compoundsagainst drug-resistant MTB (M. tuberculosis strain H37Rv), M.avium-intracellular, and M. bovis BCG. The results are shown in Table 1.Dose-response curves for the compound, designated III-50, havedemonstrated an MIC of 6.5 μg/ml against the virulent strain of M.tuberculosis, H37Rv, and 6.25 μg/ml for M. bovis BCG. Dose-responsecurves for the compound designated S-I-73 have demonstrated an MIC of3.12 μg/ml against MTB and 12.50 μ/ml against M. avium-intracellular.Tests using a compound according to Formula I having R=C₈H₁₇, Z=NH andY=—CO—O—CH₃ against M. tuberculosis, H37Rv, demonstrated an MIC of 12.5μg/ml.

Example 4 III-50 Inhibits Mycolic Acid Synthesis via a Target DifferentFrom Isoniazid

In a series of metabolic labeling experiments, the activity of a numberof lipid metabolic pathways were studied in the presence and absence ofIII-50 using two-dimensional thin-layer chromatography and phosphorimagequantification. The TLC plates were spotted with mycobacterial acidmethanolysates and developed in the first direction with petroleum ether(bp 60-80° C.):acetone (95:5, v/v, 3 times) and in the second directionwith toluene:acetone (97:3), v/v, once). Abbreviations in FIG. 2 are:Ori=origin; A=α-mycolate; B=ketomycolate; and C=ω-mycolate. The lefthand panel shows acid methanolysates from M. avium-intracellularecontrol cultures. The right hand panel shows acid methanolysates from M.avium-intracellulare cultures treated with 12.5 μg/mln-octanesulphonylacetamide (III-50).

While minor qualitative and quantitative alterations of various lipidspecies were identified, the most profound effect was noted upon mycolicacid synthesis. Reproductions of the phosphorimages (FIG. 2) demonstratethat in the presence of III-50 mycolic acids are undetectable in M.avium-intracellulare and significantly reduced in M. bovis BCG. Moreimportantly, while inhibition of mycolic acid synthesis is thought to bethe mechanism of action of isoniazid (Takayama, et al., 1972 & 1975;Quemard, et al., 1993 & 1996; and Baldock, et al.), III-50 inhibits boththe growth of M. avium-intracellulare, which is routinely isoniazidresistant (>2.5 μg/ml), and also an isoniazid (INH) resistant M.tuberculosis (>0.4 μg/ml). Thus, although both isoniazid and III-50inhibit mycolic acid synthesis, the enzymatic target of III-50 withinthe mycolic acid synthetic pathway appears to differ from that ofisoniazid.

Inhibition of mycolic acid synthesis leads to the disruption of the cellwall in mycobacteria. FIG. 3 shows electron micrographs which depictcell division of M. bovis BCG in the presence (Panel A) and absence(Panel B) of III-50. Note, in the control, the well-developed cell walland septum as the bacterium divides. In contrast, in the presence ofIII-50, there is disruption of the cell wall likely as a result ofinhibition of mycolic acid synthesis.

Preparation of a Combinatorial Library of Sulfonamide Compounds

This invention contemplates combinatorial libraries that containscompounds of Formula II:R—SO_(n)—CH═CH—CO—Y  Formula IIwhere the various symbols have the same meaning as in Formula I, exceptthat one or both of the vinyl hydrogens may be independently replaced bya group selected from alkyl, acyl, aryl, aralkyl, halogen; substitutedor unsubstituted thiol; unsubstituted or substituted amino; hydroxy, andOR′ wherein R′ is selected from the group consisting of hydrogen, alkyl,acyl, aryl aralkyl, unsubstituted or substituted amino; substituted orunsubstituted thiol; and halogen; and a linear or cyclic carbon chainoptionally interrupted with one or more heteroatom, and optionallysubstituted with one or more ═O, or ═S depending on the choice ofelectrophile in Scheme 3, or both vinyl hydrogens are replaced by alinear carbon chain to form a cyclic carbon moiety optionallyinterrupted with one or more heteroatoms, and optionally substitutedwith one or more ═O, or ═S. Typically, the groups substituted at thevinyl positions will have from 1 to 20 carbons in aggregate, and theheteroatoms will generally be selected from B, N, O, P, and S, moreusually N, O, and S. The combinatorial library of this invention mayalso include derivatives of Formula II produced by electrophilic or freeradical addition at the double bond between the acyl group and thesulfur atom.

Using the method of synthesis of vinyl sulfones 2 and 5, it is possibleto prepare collections of screenable compounds by combinatorial methods.This is illustrated in Scheme 3.

The acidity of the acetylenic hydrogen in methyl propiolate orpropiamide 3 allows its ready removal by base and reaction withelectrophiles R₁—X (halides, aldehydes, ketones, esters, etc.) to give6. Thiols R—SH can be added to 6 in the presence of mild base (i.e.,potassium or sodium carbonate, triethylamine, etc.) to give trans- ortrans/cis-mixtures of variously substituted sulfanylacylic esters oramides. Oxidation selectively to sulfoxides 8 or sulfones 9 can becarried out. Cis- and trans-isomers can be separated by crystallizationor chromatographically. By varying R₁—X and R—SH or the identity of theester or amide, a combinatorial library can be prepared.

The use of combinatorial libraries of diverse chemical compounds in drugdiscovery is well known (Moos, et al. (1993), Ann. Rep. Med. Chem., vol.28, chap. 33, pp. 315-324, Academic Press; Gordon, etal. (1994), J. Med.Chem., 37:1385-1401). Methods of screening such libraries are described,e.g., in Gordon, et al., and references cited therein. Combinatoriallibraries comprising compounds according to this invention, includingvinyl sulfones or sulfoxides prepared as described in Scheme 3, may bescreened for biological activity by any suitable screening procedure.

In an exemplary screening procedure, members of a library could betested for ability to inhibit growth of Toxoplasma gondii using a testsystem described in U.S. Pat. No. 5,614,551, incorporated herein byreference. For example, toxicity, fibroblast lysis and growth of T.gondii can be monitored in 24 well tissue culture plates containinghuman foreskin fibroblasts. Seven serial dilutions of three members ofthe library can be compared to three control wells in each 24 wellplate. Using multiple plates, large numbers of compounds from thelibrary can be screened for their effect on T. gondii. Alternatively,using fewer dilutions, more compounds may be tested in each plate.Similar screening may be performed using Mycobacterium sp. in testsystems described in U.S. Pat. No. 5,614,551 to identify compounds in acombinatorial library which affect mycobacteria. Other test systemssuitable for screening compounds in combinatorial libraries according tothis invention for their effects on other pathogens and/or neoplasticcells are readily available to the skilled worker, in view of theteachings herein.

Alternatively, the library may be screened for inhibitory effect on anenzyme such as fatty acid synthase (FAS). Suitable assay procedures aredescribed in U.S. Pat. No. 5,759,837, incorporated herein by reference,and multi-well plates may be used to perform simultaneous FAS assays inthe presence and absence of numerous members of the library to comparethe inhibitory effect of various member compounds. In anotheralternative, binding affinity of member compounds for a particularreceptor may be compared. Using a suitable assay that measures progressof a biological process, the skilled worker can readily design asuitable screening procedure to screen the combinatorial library of thisinvention for biological effect.

For purposes of clarity of understanding, the foregoing invention hasbeen described in some detail by way of illustration and example inconjunction with specific embodiments, although other aspects,advantages and modifications will be apparent to those skilled in theart to which the invention pertains. The foregoing description andexamples are intended to illustrate, but not limit the scope of theinvention. Modifications of the above-described modes for carrying outthe invention that are apparent to persons of skill in medicine,immunology, infectious diseases, pharmacology, and/or related fields areintended to be within the scope of the invention, which is limited onlyby the appended claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication, patent, or patent application was specificallyand individually indicated to be incorporated by reference.

TABLE 1 In Vitro Activities of Sulfones and Sulfoxides against M.tuberculosis MIC (ug/ml) Compound # MTB BCG MAI SI-73

3.12 12.50 HIII-50

6.25 6.25 25.00 SI-46

6.25 SI-52

6.25 HIII-206a

6.25 HIII-206b

12.50 SI-45

12.50 25.00 HIII-302

>25 SI-48

>25 HIII-56

>25 >25 DI-59

>25 JRG-I-89-2

12.5 JRG-I-89-1

25 JRG-I-89-3(mixture of cisand trans)

6.25 JRG-I-31

12.5 JRG-I-31(mixture of cisand trans)

50 JRG-I-43

50 MTB = M. tuberculosis (strain H37Rv) BCG = M. bovis MAI = M.avium-intracellulare See legend in Appendix for Methods.

1. A method of inhibiting growth of a mycobacterial cell, comprisingadministering an effective amount of a compound of formula I to thecell:R—SO_(n)-Z-CO—Y  I wherein: R is selected from the group consisting ofany alkyl group having from 6-10 carbon atoms, any unsaturatedhydrocarbon having from 6-10 carbon atoms, or any alkyl group havingfrom 6-10 carbon atoms interrupted by at least one aromatic ring; Z is—CH₂—; Y is selected from the group consisting of —NH₂, and —O—CH₃; andn is 1 or 2; and wherein, the mycobacterial cell is selected from thegroup consisting of cells of Mycobacteria tuberculosis, drug resistantM. tuberculosis, M. bovis, M. leprae, and M. paratuberculosis.
 2. Themethod of claim 1, wherein R is an alkyl group having 6-10 carbon atomsinterrupted by an aromatic ring to give ortho-, meta-, orpara-disubstitution.
 3. The method of claim 1, wherein R is a branchedalkyl group.
 4. The method of claim 1, wherein R is an n-alkyl group. 5.The method of claim 1, wherein n is
 1. 6. The method of claim 1, whereinn is
 2. 7. The method of claim 1, wherein Y is —NH₂.
 8. The method ofclaim 1, wherein: R is —(CH₂)₉—CH₃, n is 1, Z is —CH₂— and Y is —NH₂. 9.The method of claim 1, wherein: R is —(CH₂)₇—CH₃, n is 1, Z is —CH₂— andY is —NH₂.
 10. The method of claim 1, wherein: R is —(CH₂)₉—CH₃, n is 2,Z is —CH₂— and Y is —NH₂.
 11. The method of claim 1, wherein: R is—(CH₂)₇—CH₃, n is 2, Z is CH₂— and Y is —NH₂—.